The complement system is functional effector of the innate immune system consisting of a number of plasma proteins and cell membrane proteins. Activation of the complement leads to a series of protease activation cascade triggering release of cytokines and amplification of the activation cascade. The end result of the complement activation is activation of the cell-killing membrane attack complex (MAC), inflammation caused by anaphylatoxins C3a and C5a, and opsonization of pathogens. The MAC is essential for eliminating invading pathogens and damaged, necrotic, and apoptotic cells.
A delicate balance between defense against pathogen and avoidance of excess inflammation has to be achieved by complement system (Ricklin, D., et al., (2007). Nature Biotechnology, 25(11): 1265-1275). Many inflammatory, autoimmune, neurodegenerative and infectious diseases have been shown to be associated with excessive complement activity. For example, pathogenesis due to ischemia/reperfusion (I/R) injury has indicated that the complement activation leads to inflammation-induced damage in a number of diseases, including Acute Myocardial Infarction (AMI), Stroke, Hemorrhagic and Septic Shock, and complication of coronary artery bypass graft (CABG) surgery (Markiewski, M. M., et al, (2007). Am. J. Pathol. 171: 715-727). In addition, complement activation is a major contributor to a number of autoimmune diseases, including Systemic Lupus Erythematosus (Manderson, A. P., et al, (2004). Annu. Rev. Immunol. 22: 431-456), Rheumatoid Arthritis (RA), Psoriasis, and Asthma (Guo, R. F., et al, (2005). Annu. Rev. Immunol. 23: 821-852). Complement activation has also been correlated with the pathology of Alzheimer's disease (Bonifati, D. M., et al, (2007). Mol. Immunol. 44: 999-1010) and other neurodegenerative diseases such as Huntington's disease, Parkinson's disease, and age-related macular degeneration (AMD) (Gehrs, K. M., (2010). Arch. Ophthalmol., 128 (3): 249-258).
The complement system can be activated through three different pathways: the classical pathway, the alternative pathway, and the lectin pathway. All three pathways go through critical protease complexes of C3-convertase and C5-convertase that cleave complement components C3 and C5, respectively. The classical pathway is initiated by binding of C1q to antibodies IgM or IgG leading to activation of the C1 complex that cleaves complement components C2 and C4, producing C2a, C2b, C4a, and C4b. C4b and C2b then forms the classical pathway C3-convertase, which promotes cleavage of C3 into C3a and C3b. C3b then forms the C5-convertase by binding to C4bC2b (the C3-convertase). The lectin pathway is identical to the classical pathway downstream of the C3-convertase, and is activated by binding of mannose-binding lectin (MBL) to mannose residues on the pathogen surface. The MBL-associated serine proteases MASP-1 and MASP-2 can then cleave C4 and C2 to form the same C3-convertase as in the classical pathway. Unlike the classical and the lectin pathways that are specific immune responses requiring antigens, the alternative pathway is a non-specific immune response that is continuously active at a low level. Spontaneously hydrolysis of C3 leads to C3a and C3b. C3b can bind Factor B and then cleave Factor B to Ba and Bb with facilitation of factor D. The C3bBb complex which can be stabilized by binding of Factor P (Properdin) is the C3-convertase of the alternative pathway that cleaves C3 to C3a and C3b. C3b can join the C3bBb complex to form C3bBbC3b complex that is the C5-convertase of the alternative pathway. The C5-convertases from all three pathways can cleave C5 to C5a and C5b. The C5b then recruits and assembles C6, C7, C7, C8 and multiple C9 molecules to assemble the MAC. This creates a hole or pore in the membrane that can kill or damage the pathogen or cell. The complement system is tightly regulated by two mechanisms: decay accelerating activity (DAA) and cofactor activity (CA). DAA refers to the ability to promoting dissociation of the C3-convertase or C5-convertase. CA refers to the ability of facilitating Factor I to cleave C3b or C4b to inactive fragments. For a review the complement system see Wagner, E., et al., (2010), Nat. Rev. Drug Discov., 9(1): 43-56.
Human Complement Receptor type 1 (CR1) is the only complement regulator that has DAA for the both classical and alternative C3-convertases and C5-convertases and CA for C3b and C4b, and therefore has generated interest in therapeutic applications (Krych-Goldberg, M., et al, (2001), Immunological Reviews, 180: 112-122). A naturally occurred soluble human CR1 (sCR1) lacking the transmembrane and the intracellular domain have been shown to inhibit the complement system in vitro and various in vivo animal studies (Mollnes, T. E., et al, (2006), Molecular Immunology, 43: 107-121). sCR1 has also been tested in human clinical trials to reduce tissue damage in myocardial infarction (Perry, G. J., et al, (1998), J. Am. Coll. Cardiol., 31: 41 1A), adult respiratory distress syndrome (Zimmerman, J. L., et al, (2000), Crit. Care. Med., 28(9): 3149-3154), and lung transplantation (Zamora, M. R., et al, (1999), Chest, 116: 46s). It has been found safe, non-immunogenic, and efficacious in term of inhibiting complement activities in vivo. However, the molecular structure makes sCR1 difficult to produce as a therapeutic agent. Deletion mutagenesis has identified that the first 3 SCRs (SCR1-3) was sufficient to convey the DAA for the C3-convertases but not the CA for C3b and C4b (Krych-Goldberg, M., (1999), J. Bio. Chem., 274(44): 31160-31168). Similar to CR1, complement regulatory proteins DAF, MCP, Factor H, and C4BP contain a number of SCRs where the binding sites of C3b or C4b and the active sites for complement inhibitions have been mapped (Makrides, S. C., (1998), Pharmacological Reviews, 50 (1): 59-87). Soluble forms of MCP, DAF, and Protectin have been produced and shown to be effective to inhibit complement in vitro and various animal models (Wagner, E., et al., (2010), Nat. Rev. Drug Discov., 9(1): 43-56). However, they have relatively low potencies and short half-lives in vivo.
Vascular endothelial growth factor (VEGF) is one of the most important proteins that promote angiogenesis, which is a tightly regulated process of developing new blood vessels from a pre-existing vascular network (Ferrara, N., (2004), Endocrine Reviews, 25(4): 581-611). Angiogenesis is required during development and normal physiological processes such as wound healing, and is also involved in a number of disease pathogenesis, including AMD, RA, Diabetic Retinopathy, tumor growth and metastasis. Inhibition of angiogenesis has been shown to be effective in therapeutic applications.
The VEGF pathway and complement pathway both contribute to the formation of diseases with similar etiologies. Therefore there is a need for the development of therapeutic agents that target both the VEGF pathway and complement pathway. Provided herein are fusion proteins that inhibit activation of both the complement pathway and the VEGF pathway. Fusion proteins of the present invention can be used as therapeutic agent to for use in treatment of complement- and VEGF-related diseases.